Identification and characterization of lymphocyte subpopulations

Identification and Characterization

of Lymphocyte Subpopulations

By Dick L. Robbins and M. Eric Gershwin A pathogenic agent may cause signs of illness only when modified by the presence of antibody. Clemens von Pirquet, 1902

T

HE WELL-KNOWN OBSERVATIONS of Jenner, Pasteur, and von Pirquet of natural and iatrogenic induced pathology have been the cornerstone for developing our enormous cataloging of immune mechanisms, development of vaccines, isolation of antibodies, and use of both immunostimulatory and immunosuppressive agents in efforts to achieve optimal balances between immunologic status and acquired disease. Indeed, the majority of immunologic therapeutic modalities currently employed in clinical medicine were long idiosmallpox vaccine, before matic, e.g., mechanistic issues could be entertained. In fact, studies of immune many elementary mechanisms could not be seriously undertaken until a series of cascading fortuitous observations were made in mice, chickens, and human congenital immune deficiency states. These investigations led to the realization that the immune system can be subdivided and characterized into two major components, a thymic-derived (T cell) and a bursa-derived (B cell) pathway. This dual component concept, in which thymic-derived or cell-mediated immunity is distinguished from bursa-derived or humoral immunity, has gained widespread usage throughout all phases of developmental and comparative immunology, and it is now in vogue to study and classify qualitative alterations of these two major compartments in genetic, immunologic, and neoplastic disease. Because of the omniscent nature of this classification, we hope to place in perspective current concepts of lymphoid subpopulations, the experimental basis upon which these thoughts were born (or hatched!), as well as to discuss lymphoid surface marker and separation procedures from a phylogenetic and ontogenetic viewpoint and, finally, their alterations in specific human disease. Because of the growing awareness of the integral relationships and interactions between lymphoid subpopulations in congenital and acquired dysimmunity, the thesis will be expanded to permit a major thrust at discussing Seminars in Arthritis and Rheumatism, Vol. 7. No. 4 (May). 1978

positive and negative features of immune regulation in immunopathology. EXPERIMENTAL

WORK

IN ANIMALS

Although there have been many critical observations relative to the discovery that the immune system of vertebrates can be separated into a cell-mediated and a humoral component, the earliest serious experiments were derived from an accidental discovery in chickens.lw3 In 1956, Glick and his co-workers at the University of Wisconsin were studying the role of the bursa of Fabricius, an organ located just anterior to the cloaca of the chicken at the distal end of the gastrointestinal tract, on growth and development. They found, much to their disappointment, that bursectomy did not alter subsequent growth and weight gain. During the course of these studies, several of these neonatally bursectomized chickens were accidentally provided to Chang, another investigator. Chang used these birds, as well as normal chickens, in a student demonstration attempting to elicit antibody response to the antigen Salmonella typhimurium. A number of the neonatally bursectomized chickens were unable to develop significant antibody titers following immunization. This relationship between neonatal bursectomy and absence of antibody production was quickly grasped by Glick and Chang.le4 Subsequently, this observation formed the vanguard for a large number of studies that ultimately demonstrated the critical role of the bursa on the development of antibody formation.3-s Specifically, it was demonstrated that From the Section of Rheumatology~CIinical Immunology, University of California School of Medicine, Davis, Calif: Supported by funds received from the Kroc Foundation. M. Eric Gershwin is recipient of Research Career Development AwardAIOO193. Dick L. Robbins, M.D.: Assistant Professor of Medicine; M. Eric Gershwin, M.D.: Associate Professor of Medicine. Section of Rheumatology-Clinical Immunology, University of Calijbrnia School of Medicine, David, Calif: Address for reprint requests to: M. Eric Gershwin, M.D.. TB 171, University of California School of Medicine, Davis, Calif: 95616. 0 I978 by Grune & Stratton, Inc. ISSN 0049-0172. ISSN 0049-0172/78/0704-00#1$05.00/0 245

246

removal of the bursa immediately after hatching virtually eliminated production of specific antibody.l In contrast, removal of the bursa later in life did not significantly impair antibody production. Neonatally bursectomized chickens, although having markedly reduced serum immunoglobulins and absence of plasma cells, had relatively normal numbers of small lymphocytes in both peripheral blood and spleen and were also capable of normal allograft rejection.‘-lo Even repeated antigenic stimulation of neonatally bursectomized chickens failed to elicit antibody production. Similarly, neonatal bursectomy blocked development of splenic germinal centers.“-” This paradox between bursectomy and failure of antibody production but retention of normal cell-mediated immunity (ability to reject allografts) was resolved by Cooper and co-workers. They found that thymectomized irradiated newly hatched chickens failed to develop cellmediated immunity but that plasma cells, germinal centers, immunoglobulin levels, and circulating antibody responses all developed normally.“-4.x,” On the other hand, bursectomy and irradiation in newly hatched chickens blocked development of larger lymphocytes, germinal centers, and plasma cells and resulted in agammaglobulinemia and deficient circulating antithat the body levels. ‘o-‘4 It became apparent thymus is necessary for differentiation of the small lymphocytes responsible for cellmediated immunity and the bursa for the development of humoral immunity. 1m6.‘2m’(i Study of the association of bursa development and B-cell ontogeny continues to be useful for understanding lymphocyte maturation. Bursectomy of chickens at serial ages after hatching has revealed an age-dependent relationship for development of specific immunoglobulin classes.” For example, while neonatal bursectomy results in total inhibition of antibody production of all immunoglobulin classes, bursectomy performed shortly after hatching selectively inhibits IgG and IgA but not IgM antibody responses. This influence of the bursa on immunoglobulin appearance suggests that IgM ontogenetically precedes IgG and IgA and that IgM-bearing B cells precede IgG- and IgAbearing B cells. This progression of B-cell maturation has been demonstrated in other animals; the proposed order is IgM, IgG, and finally

ROBBINS

AND

GERSHWIN

IgA. Ix As might be predicted from these observations, a B-CellLdeprived animal can be produced by continued treatment of newborn animals with anti-p antibody (antibody against the heavy chain components of IgM).“.” At approximately the same time that bursectomies were arousing interest, it was discovered that neonatal thymectomy of mice and rabbits resulted in severe impairment of allograft rejection.“‘-2” In contrast to neonatal bursectomy, neonatal thymectomy of mice depletes both small and medium lymphocytes and markedly diminishes delayed hypersensitivity; however, adequate plasma cells and total immunoglobulin levels remain essentially at presurgical values. ” This dichotomy between the effects of thymectomy and the effects of bursectomy contributed enormously to the appreciation that each controlled a separate limb of the immune system. Thus the concept of cellmediated versus humoral immunity was established”.‘“,‘“,‘” (Fig. 1). It was also apparent that in addition to its influence on cell-mediated immunity, thymectomy also resulted in defective antibody responses when experimentally manipulated animals were exposed to antigens such as sheep red blood cells (SRBC) and certain bacterial antigens.g”- 2+ However, antibody responses to other antigens (e.g., pneumococcal polysaccharide) remained normal in thymectomized animals.“2P2” Subsequent work demonstrated that the T-cell system frequently “cooperates” with cells of bursa origin to produce normal antibody formation.2”-2X Thus T cells are necessary for a normal B-cell response to certain antigens known as thymic-dependent

Fig. 1.

Schematic

munity.

Note

tion

the

of

significant

representation

that although cellular

functional

and

there humoral

of the ontogeny

is independent components.

interrelationships.

of im-

differentiathere

are

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247

SUBPOPULATIONS

antigens but not for other (thymic independent) antigens. This latter group of antigens, which include pneumococcal polysaccharide, levan, lipopolysaccharide, DNP, Ficoll, polyvinyl, pyrrolidone, and poly I . poly C, is both unique and interesting because of the polymeric structures.2gs”0 While they elicit antibody response without T-cell help, it is only IgM, and the immunocompetent cells stimulated develop no immunologic memory and can be easily made tolerant.“‘.:3” Further support of the contrasting influences of the thymus and bursa is derived from experiments illustrating that the deep cortical regions of the lymph node were most profoundly effected by neonatal thymectomy, whereas germinal centers, outlying cortical areas, plasma cells, and medullary cords were affected by bursectomy.a-6.33 Similarly, neonatal thymectomy of mice, much as in untreated patients with DiGeorge syndrome, results in characteristic wasting and runting phenomena.3”*3” Moreover, the wasting seems to be directly related to the degree of development of the peripheral lymphoid tissue at the time of birth. Mice kept in germ-free environments develop less runting and wasting than animals undergoing neonatal thymectomy and thence exposed to pathogens.33-3s Finally, as might be predicted, thymectomy performed at 2-4 wk of age is much less detrimental than when done at birth.33-3X Further experiments demonstrated that the ability to reject allografts or initiate graftversus-host reactions, both abrogated by neonatal thymectomy, can be restored by thymus grafting.“x.3g Syngeneic tissue was more effective than an allogeneic transplant in reconstituting immune function; transplants mismatched at the H-2 locus usually failed to re-

Moreover, in many instances constitute.lO-*” allogeneic grafts mismatched at the H-2 locus induced lethal reactions.43 These observations were likewise true when peripheral lymphoid tissue such as lymph node and spleen was used to reconstitute neonatally thymectomized mice.42-4~

Continued study of inability of neonatally thymectomized mice to participate in cellmediated immunity and neonatally bursectomized chickens to produce immunoglobulins has led to the identification of the origin, site of differentiation, and recirculation patterns of lymphocytes. Hemopoietic stem cells arising from the yolk sac, liver, or bone marrow migrate, possibly under the influence of a humoral factor, to the primary lymphoid organs, the thymus or bursa. These stem cells differentiate within a local epithelial-reticular structure to form populations having specific surface marker characteristics of T or B cells.46-4g Thymusderived cells then circulate to populate the thymus-dependent areas of lymphoid tissues, whereas bursa-derived cells populate the thymus-independent areas of lymph nodes (Fig. 1). Although the majority of experimental data used to separate cell-mediated from humoral immunity are derived from work on chickens and mammals, the two-component concept of immunity, i.e., cell-mediated and humoral immunity, can be illustrated using phylogenetic pathways (Table 1).50--53 Responsiveness to foreign material is one of the most fundamental and basic processes of life and can be demonstrated even in that primitive one-cell organism the amoeba. Development, however, of specialized immune apparatus appears to begin with the invertebrates (Table 1). Earthworms and caterpillars, for example,

Table 1. Phylogeny of Immunity Cell-mediated lmmumty

SpeCWZS Earthworm

+ Illmired)

Hagfish

+ (Ilmlfed)

Horned shark

+ (more extensive

Antibody

Plasma

Two-Component

ReSpOnSe

Thymus

Cells

Lymph Nodes

_

_

_

_

+

_

Prlmltive lymphoid

hemoblast

_in antenor kadney

than above)

-

+ (weak but present)

Prlmltive pharyngeal

epithelial

structure

Leopard shark

+ (distinct thymus)

+

+

+

_

Frog

+

+

+

+

_

Alligator

+

+

+

+

Chicken

+

+

+

+

_

Mice

+

+

+

+

+

248 demonstrate the capacity to recognize specific antigenic determinants and can be induced to show immunologic memory.53 Moreover, although they do not possess central lymphoid organs, there is evidence for both rudimentary cell-mediated and humoral components of immunity.“‘-“” For example, earthworms within their coelomic fluid and hemolymph contain broad-reacting agglutinins to foreign erythrocytes and bacteria. Although these agglutinins are broadly reactive and do not migrate electrophoretically in the same region as gammaglobulins, they are nonetheless bacteriostatic and occasionally bactericidal.“s-“7 Although earthworms do not possess a thymus gland, they are nonetheless capable of slowly rejecting of allografts.“6 Although the interrelationships these primitive systems are not thoroughly understood, it is nevertheless apparent that they are independent, but interrelated, systems. Primitive separation of cell-mediated and humoral pathways exist far below bird or mammalian phyla (Table 1). The division of immunologic responsiveness into cell-mediated and humoral components has obviously been successful because nature has continued to conserve and differentiate within these major divisions, and the two-component concept of immunity can be illustrated in all higher forms of life. The separation of cell-mediated from humoral immunity is noted even in the marsupial mammals of Australia and New Zealand, animals long separated from the mainstream of evolution. As one ascends the animal kingdom from invertebrates to the true vertebrates, an anatomically distinct thymus gland and specific immunoglobulinlike proteins appear. Both T and B cell responses can be induced in the lowest vertebrate studied, the California hagfish (cyclostone). ,57 Moreover, probably secondary to the appearance of a thymus gland, cellular immunity, immunologic memory, and delayed hypersensitivity can be found. Perhaps as a corollary to development of these sophisticated features, it is intriguing to comment on the phylogenetic appearance of lymphoid malignancy and autoimmune disease as immunologic diversification and sophistication becomes evident. For example, lymphomas of frogs and chronic biliary cirrhosis of alligators are well accepted albeit infrequently discussed ailments of

ROBBINS

AND GERSHWIN

these species.“‘.“” Similarly, fish, following exposure to either radiation or carcinogens, respond in a fashion similar to humans. It is an unfortunate highlight of 20th century technology that development of tumors in fish can be used to monitor pollution of our ponds and streams.6o*6’ Study of the phylogeny of the immune response therefore illustrates that appearance of the thymus correlates with development of delayed hypersensitivity and development of the bursa (or its equivalent) with antibody formation. Ontogenetic considerations of immunity likewise illustrate the two-component nature of immunity. From using the chicken as a model it is clear that the central lymphoid organs, the thymus and bursa, act as sites for the differentiation of immunocompetent cells.‘-’ The products of the bursa of Fabricius are small lymphocytes known as bursacytes or B cells.” In contrast, the products of the thymus gland are small lymphocytes known as thymocytes or T cells.4-6 These central lymphoid organs have a similar critical feature, namely, organization around a reticular epithelial framework. It is now known that the factors required for adequate differentiation of these bursacytes and thymocytes into mature immunocompetent cells are derived from and are present in these reticuloepithelial frameworks.“2~6” The critical significance of the epithelial framework is illustrated by recent demonstrations of a selective T-cell deficiency state secondary to human congenital dysgenesis of thymic epithelial cells. EXPERIMENTS

OF NATURE

It has long been characteristic of clinical medicine that our knowledge and learning experience is heavily derived from observations of rare congenital states and diseases that affect only a small minority of patients.“7 This is well illustrated in clinical immunology because of the overwhelming contributions of pediatric immunology and study of patients with myeloma towards our present state of the art. In particular, in 1957 Bing and Plumb noted the association of hypergammaglobulinemia and plasmacytosis in several patients, leading to the suggestion that plasma cells were the source of immunoglobulins. This hypothesis was supported by other observations, including the association in a patient with subacute bacterial

LYMPHOCYTE

SUBPOPULATIONS

endocarditis of elevated gamma globulins and extreme plasmacytosis.68-7’ Earlier investigators had proposed that lymphocytes were the sole producers of antibody, but it became necessary to alter this concept as it became evident that the major source of antibody production was the plasma cell. Nonetheless, several more years were required before the relationship between differentiation of B cells into plasma cells and their subsequent production of antibody was appreciated.6g-74 The relationship between plasma cells and immunoglobulin production, however, became better illustrated and studied in another experiment of nature, namely, multiple myeloma. It was well known that patients with this disease had an extensive plasma cell infiltration of bone marrow and parenchymal organs. Because myeloma is also characterized by an elevation of gamma globulins, the association between plasma cells and antibodies became thus firmly established. Subsequent demonstrations revealed that the immunoglobulin elevation noted in multiple myeloma was extremely homogeneous and that these immunoglobulins were immunochemically related to all the others and to normal gamma globulins but were immunoat the same time unique chemically.7”-77 This observation laid the foundation for the developing field of molecular immunology. The experiments in chickens and mice noted above proved that the thymus gland was essential for development of cell-mediated immunity as well as production of antibody to thymic-dependent antigens. Nevertheless, the role of the human thymus gland remained to be resolved and was controversial. Many groups, for example, considered it little more than a vestige. Although there had been associations between human thymomas and immunodeficiency, it was the intensive study of select congenital human deficiencies that led directly to appreciating the significant nature of the thymus. DiGeorge syndrome, in which patients are born without a thymus gland, is one such immunodeficiency syndrome.78*7g Clinically, the absence, either partial or complete, of T cells is manifested by failure to develop either delayed or contact allergic reactions and inability to reject skin grafts. Because the B-cell pathway of lymphocyte development is normal, immuno-

249

globulin levels and development of antibodies to thymic-independent antigens are norma1.7g For example, in one patient studied, 85% of the circulating lymphocytes were B cells, as compared to only 20%30% in normals.7g The identification of this syndrome as secondary to a failure of T-cell development, itself due to thymic agenesis, suggested that thymic transplants might effectively replace the deficiency. This has occurred on several occasions,7g~so and DiGeorge patients have developed T-cell function following thymic transplant much more rapidly than expected. In contrast to the selective T-cell deficiency of DiGeorge syndrome, there exists a selective B-cell deficiency characterized by absence of plasma cells and agammaglobulinemia.x’-X1 Patients with this deficiency have a normal thymus gland and are thus capable of delayed and contact hypersensitivity as well as rejection of skin allografts. Nonetheless, they are unable to make significant antibody responses to either thymic-dependent or -independent antigens.84-86 Agammaglobulinemia therefore appears to result from a specific defect in the development of B cells. Since the equivalent of the bursa of Fabricius in mammals has not been definitively demonstrated, the site of this abnormality remains elusive. However, as will be noted below, unlike DiGeorge syndrome, selective agammaglobulinemia appears to be due not to an embryologic defect but rather to development and appearance of a unique lymphocyte population that chronically suppresses the Blymphocyte population.s5~s6 There are a large number of other congenital immunologic abnormalities of humans. These have been extensively reviewed elsewhere and will not be individually discussed here. However, the several phenotypic variations that exist in human immunodeficiencies are critical to our understanding of the interrelationships in the development of both cell-mediated and humoral immunity and have provided investigators with fertile experimental bases for immunotherapy. Because of the selective nature of several of these congenital deficiencies, the effects of thymic, fetal liver, and bone marrow transplants can be adequately observed. In addition to the use of these experimental models to study the relationship between cellmediated and humoral immunity, clinical

250

observations of infectious disease in patients with multiple myeloma and Hodgkin disease have likewise disclosed a similar dichotomy based upon altered susceptibility to infectious disease.“7,XH For example, patients with multiple myeloma experience recurrent bacterial infections with high-grade bacterial pathogens, such as pneumococcus and Hemophilus; they do not, however, unless influenced by chemotherapy or until terminally ill, have major increased morbidity from either viral or fungal infectionsxx In contrast, patients with Hodgkin disease demonstrate markedly increased susceptibility to viral, fungal, and acid-fast baccillus infections.x7 Both of these facts are similar to the increased susceptibility to bacterial infections in patients with agammaglobulinemia and to viral diseases in patients with DiGeorge syndrome. It is proposed that patients with Hodgkin disease are more susceptible to virus and fungi because of development of defects in cell-mediated immunity. In comparison, patients with multiple myeloma have difficulty with high-grade encapsulated bacterial pathogens because of the critical role of the B-cell pathway in responding to these infections.x7-xY Thus the separation of the immune system into thymic- and bursaderived components is of more than academic interest. Indeed, it remains one of the most fundamental concepts in understanding host relationships to infectious disease, autoimmunity, and neoplasia. ANIMAL

MODELS

OF CONGENITAL

DEFICIENCY

IMMUNE

STATES Fig. 2.

Unfortunately, congenital immune deficiency states of animals were until recently either unknown or ignored. In the past several years, however, a number of unique animal models with particular relevance to human disease have been described. In 1966 hereditary absence of hair was discovered as a mutation of mice in Scotland. Such mice were also noteworthy for failure to thrive, development of diarrhea, and death from a runting illness.“” In 1968, serial sections of the mediastina of these animals, known as nude mice, failed to reveal evidence of a thymus gland (Fig. 2). These dual traits of hairlessness and athymia are inherited in a simple Mendelian recessive pattern with complete penetrance. As one might expect, nude mice lack all evi-

(A) Congenitally

this mouse munity,

lacks

it is capable

and xenografts. derived

of only

10” human to increase

munity.

(B)

free

must

conditions,

defects plenic

which

of

of both mouse.

of the asplenic

be housed and

mass

severe B-cell

Note deformed

of both

present

approximately carcinoma

with

an

under

qualitative

mouse

and (C)

will

of host imderived

mouse.

pathogen-free

function.

on this

cells. Tumor

asplenic

imallo-

6 wk earlier

interference

athymic-asplenic

a nude

has T-

tumor

Because

cell-mediated

acceptance

in size without

Congenitally

inbreeding

large

a transplant endometrial

continue

animal,

the

from

(nude1 mouse.

and therefore

of long-term

Note

mouse,

from

athymic

a thymus

This

or gerrn-

quantitative

Hereditarily

hind legs, a phenotypic

as-

marker

mouse.

dence of cell-mediated immunity, including ability to reject allografts, development of contact hypersensitivity, and responsiveness to thymic-dependent antigens.!“-“:’ In contrast, nude mice demonstrate responsiveness to thymic-independent antigens and have signifi-

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cant levels of serum IgG and IgM.S3 However, nude mice have markedly depressed levels of serum IgA.g2-g” This selective deficiency of IgA is unique among murine systems and parallels similar observations of depressed IgA levels following neonatal thymectomy of mice and rabbits.g”,“” These observations have suggested that IgA production is in part under thymic influence.g7 Because nude mice can be readily studied they lend themselves to immediate attempts at immune reconstitution.g8 As one might expect, they are analogous to humans with DiGeorge syndrome and immune reconstitution can be accomplished with thymic transplants.g3 Several other immunologically mutant mice have been described. One line with particular clinical relevance is the hereditary absence of the spleen in mice (Fig. 2). Such mice are both born without a spleen and can be phenotypically identified by associated abnormalities of the hind limb.gg These traits are inherited as autosomal dominant, permitting the raising of large numbers of mice. Although the hereditary absence of the spleen is not as immunologically compromising as thymic agenesis, affected mice are useful for studying the relationship between the spleen and development of immunocompetence. For example, hereditarily asplenic mice have delayed thymic maturation, probably secondary to the influence of the spleen on T-cell ontogeny.gg.“‘o As one might expect from clinical observations, hereditarily asplenic mice have an increased susceptibility to high-grade encapsulated bacterial pathogens, e.g., pneumococcus. As an interesting sidelight, the mating of nude with asplenic mice under pathogen-free conditions has produced congenitally athymic-asplenic mice with extensive derangements of both T- and B-cell function. In addition to mutant mice, there is a wellknown severe combined immune deficiency of Arabian foals. This trait, similar to that of nude mice, is inherited in an autosomal recessive pattern with complete penetrance; affected foals lack evidence of both cell-mediated and humoral immunity.‘O’ Such immunologically deficient horses appear much like severe combined immunodeficiency (SCID) of humans.“’ There is virtual absence of the thymus gland, profound lymphopenia, absence of plasma cells, and agammaglobulinemia,‘OL but

in contrast to the low levels of adenosine deaminase in some humans with SCID, affected foals have normal levels.‘03 More recently, an acquired agammaglobulinemia of inbred chickens has been described. Such birds appear normal at birth and then progressively lose either IgG or IgM production.‘O* This development of selective agammaglobulinemia is non-sex-linked, and its inheritance appears polygenic. In many ways it appears analogous to acquired agammaglobulinemia of humans. lo5 This bird model is now receiving considerable attention because chickens lend themselves to a variety of readily performed surgical procedures following hatching because of the ready accessibility of the bursa, thymus, and spleen and the absence of lymph nodes. Moreover, these chickens, in contrast to congenitally athymic (nude) mice or Arabian foals, are genetically highly inbred and thus lend themselves to studies of cell transfer and immune reconstitution without the problems of allogeneic interference. IDENTIFICATION

AND

PROPERTIES

OF T CELLS

Lymphocytes derived from the thymus (T cells) are responsible, as noted above, for cellmediated responses. These include the initiation of delayed allergic reactions, including solid allograft rejection, graft-versus-host disease, contact sensitivity, and recognition of immunologic responsiveness (and possibly immunosurveillance against neoplasia).“‘“-‘O” T cells also contribute a major specific component in the body’s defense against a variety of pathogens, including facultative intracellular bacterial pathogens, viruses, and fungi. IoXMorphologically, they consist of the cell population in the deep cortical areas of lymph nodes following antigenic stimulation (Fig. 1; Table 2). Qualitative and quantitative aspects of T-cell function can be evaluated in a number of ways both in vitro and in vivo. By far the simplest clinical method involves measuring delayed hypersensitivity via skin testing.‘0g-“6 Skin testing remains the most important clinical assessment in evaluating the status of the cellular immune system. This important relationship was noted by Jenner in 1801 when he described the “disposition to sudden cuticular inflammation” following the injection of cowpox into the skin of

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252

Table 2. Characteristics

of T Cells

Initiated delayed hypersensitivity Responsible for graft-versus-host Development

Form spontaneous Transformed

reactio”

of contact allergy

wth

rosettes mth sheep erythrocytes phytohemagglutmln.

concanavalin

A. and

Sodium perIodate Respond an vitro to mitomycin-treated Responsible

or irradiated allogeneic cells

for rejection of allografts

as both helper and suppressor cells

Functw”

patients previously infected with cowpox or of delayed This concept smallpox. hypersensitivity was further expanded by the astute observations of Koch following injection of extracts of tubercle bacilli into the skin of guinea pigs. These injections, when performed in tuberculin guinea pigs, caused the appearance of gross inflammation within l-2 days. In contrast, injection of tubercle bacilli extracts into normal guinea pigs did not produce a reaction for lo-16 days. ‘I’ This relationship between previous exposure and latency periods for responsiveness initiated the concept of delayed hypersensitivity in the tuberculin test.“‘,“” Hypersensitivity to tuberculin could be readily separated from an Arthus or immediate reaction based upon the contrasting ability of lymphocytes and sera to transfer the response. “‘-‘13 For example, Arthus reactions are mediated by IgE and can be passively transferred from an allergic to a control animal by sera, not cells. In contrast, transfer of tuberculin sensitivity depends on passage of lymphocytes.‘13.“4 Table 3. Commonly Used Skin Tests for Delayed Hypersensitivity

in Man

Test Extract

DlS‘33se Tuberculosis

Purified protein derivatwe

Leprosy

Lepromin Isuspension of leprosy bacteria)

(PPD)

Brucellosis

Brucellin (filtrate of Bruce/la abortus or melitensis Brucellergen

(bacterial nucleoprotem)

Glanders

Mallium

(filtrate of glanders bacillus

Tularermta

Prote,n extract of Pasteurella

Mumps

Kllled virus

Psittaccws

Kllled virus

culture) tularensis

Cat scratch fever

Extract from affected lymph nodes

Candidiosis

Candida albicans

Coccidioidomycosis

Coccadioidm

Histoplasmosis

Histoplasmm

Dermatomycosls

Trvzhophyton

Lymphogranuloma “e”ere”m

Extract of yolk sac of mfected egg

Lelshmanlasls

Culture extract

Hydatud disease

Casont antbgen lhydatid cyst flu(d)

AND GERSHWIN

Presently there are more than 20 antigens that may be used for skin testing in humans (Table 3). These include, most commonly, Candida, mumps, PPD, SK-SD, and trichophytin. Other antigens may be used for specific diagnostic purposes and if anergy is considered. If anergy is suspected in a patient and if it is of clinical importance, a better evaluation than applying a battery of tests can be obtained by attempting with dinitrochlorobenzene sensitization DNCB in acetone is applied to the (DNCB).“’ upper arm of the patient and the area is allowed to dry. Approximately 2 wk thereafter, DNCB, in varying concentrations, is reapplied and the development of induration is serially observed and quantitated. There are multiple factors and clinical situadelayed in diminished which tions hypersensitivity is manifest (Table 4). The common denominator of these pathophysiologic states is a defect in thymic-derived (or cellmediated) immunity. There are, nevertheless, major differences that exist, even within normal volunteers, in ability to induce and elicit delayed hypersensitivity. These factors include genetic cellinfluence, sex, and age. 111-‘11Furthermore, mediated immunity is present very early in life, but ability to respond to select antigens (e.g., PPD) may not be present in the newborn period. Moreover, viral infections, including that resulting from vaccination with attenuated measles virus, may transiently suppress delayed hypersensitivity.“*-76 There are several methods by which lymphocyte function can be quantitatively assessed in vitro. In particular, the selective stimulation and blast transformation of either T or B cells by mitogenic agents has proven to be a major tool for the study and recognition of antigenic activation, separation of lymphoid subpopulations, and biochemical signals.‘“-“! It has been demonstrated, for example, that the lectins extracted from the plants Phaseolus vufgaris and Concanavalia ensiformis, namely, phytohemagglutinin and concanavalin A, specifically stimulate T lymphocytes.“!’ In contrast, other mitogens, including PPD and LPS, selectively stimulate B cells. ‘I9 Although the degree of transformation can be quantitated by morphologic counting of blast cells, the more reliable and objective means is to measure tritiated thymidine incorporation, a direct parameter of DNA synthesis.‘“”

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DelayedHyFrsensitivity in Man

Table 4.

Causes of Diminished

state

Ca”*a*

Congenital DiGeorga syndrome

T cell

Wwcoft-Aldrich

?

syndrome

BgB”es15

lmpawed T cell mat”,atmn

Severe combined immunodefic,ency

Stem cell defect

Ataxaa telangrectas~a

Thymlc hypogenaois

lmmunodeficiency

? Suppressor T cells

with thymoma

IgA deficiency

Relationship

between thymus

and IgA

Acquired Infectmu*

IViral) Lymphocytotoxic

Measles

antibodies

to T cell* Suppressor c*,,s

influenza I,lfectlo”*

mo”o”“cleos,s

Ant~genlc competeion V,rsl infestation

Mumps

end/o,

of T cells

monocyter

Other

tkpatit,s Other

(BFlCte,lall Tuberculosis

N”trltlon

Lepros”

Effects Of suppressor celk Effects Of lymphocyte recirc”lation

patterns

(Fungall Cwadmidomycosis

N”trit,anal

C,YPiOCOCCUS

Effects of supp,esscJ, c*11* Effects at lymphocyte reC,,c”latlOn pattern* Other

Iltl,,l”“0S”pp,*~bl0” Radtation

Ma,,gnant Hodgkin dwase

Raplacement proliferation

Leukemia

Development

Other

OlhW,

by unregulated of B cells of suppressor

cell pOp”latl0”

Metabolic Hypothyroidism “eamm

C defic,ency

bon deticiency P,c.tein defic,ency

Autmmmune

disease

Prtmary bihary urrhos6

Lymphocytotoxic

antibodies

for T cell Rheumatoid

arthritis

Dwslopment

of sUppressOr

lymphocvte Systemic lupus erythematosus

Effects on lymphocyte

SjGgren syndrome

? ln”“ence

of med,ato,s

7 Influence

of meddlators

,eci,c”lat,on

patients

Other

ldiopafhlc dwases Sarcoodosis

I Suppressor celk Other

Similarly, T cells have been found to be the principal responding population in mixed lymphocyte reactions and are the major populations recognizing histocompatibility differences.‘21*‘22 Stimulation by foreign cells

therefore can be used both as a measure of Tcell function as well as a means of distinguishing major genetic differences between individuals. Finally, mixed lymphocyte reactions also generate cytotoxic lymphocytes.‘22 There are several soluble mediators produced by T cells, B cells, and monocytes that reflect in large measure the status of cellular immunity.123-‘26 These include mediators affecting macrophages, such as migration inhibition factor (MIF) and macrophage activating factor (MAF); mediators affecting lymphocytes, such as mitogenic factors, helping and suppressing factors; mediators affecting basophils and eosinophils; and miscellaneous others, including interferon, skin reaction factors, chemotactic factor, and the family of lymphotoxins.‘23-‘26 Discussion of these is beyond the scope of this review, but they represent an exciting field that is becoming of increasing importance in understanding lymphocyte interactions. In addition to these functional parameters of T-cell function, there are a number of unique surface marker properties distinctive for T cells. One of the most important arose from the discovery that human T cells bind to sheep red blood cells (SRBC). ‘27*128Approximately 60%70% of human peripheral blood lymphocytes bind to SRBC; such lymphocytes are T cells. The method of inducing and measuring this very important marker is, briefly, as follows (Fig. 4): Lymphocytes are mixed with packed SRBC in undiluted heat-inactivated SRBC-absorbed fetal calf serum. This mixture is centrifuged and incubated at 37” C and thence at 4°C. The lymphocyte-SRBC mixture is then carefully resuspended, and the number of lymphocytes binding three or more SRBC are enumerated in a hemocytometer. The binding of SRBC to T cells is quite weak, and rosettes can be easily destroyed by agitation. However, the inclusion of fetal calf serum appears to stabilize rosettes and make them considerably less fragile. Moreover, rosettes are formed only around living cells, and such formation can be markedly decreased by EDTA, metabolic inhibitors, and elevated levels of cyclic AMP; treatment of SRBC or T cells with trypsin likewise markedly decreases rosette formation.‘27-130In contrast, treatment of SRBC with either papain or neuraminidase increases rosette formation, perhaps by stabilizing rosettes or by exposing hidden SRBC receptors on

254

other lymphoid subpopulations, e.g., B cells.‘27-‘“’ The fact that E rosette-forming lymphocytes are indeed T cells can be demonstrated by their responsiveness in mixed lymphocyte reactions, their involvement in delayed hypersensitivity, their responsiveness to phytohemagglutinin and concanavalin A, and their inhibition by antithymocyte sera. There are several other surface receptors characteristic of human T cells. In particular, the formation of rosettes with SRBC has been modified in a similar assay known as active rosette formation. In this system, rosette formation occurs without the presence of fetal calf serum, entirely at 37”C, and particularly within a very rigid relationship of SRBC-tolymphocyte ratio. Although approximately 60%-70% of cells are rosette-forming cells in normal human peripheral blood, only approximately one-third of such cells (20% of peripheral blood lymphocytes) form active rosettes.“” It has been suggested that the number

ROBBINS

AND GERSHWIN

of active rosette-forming cells is a more sensitive monitor of cell-mediated immunity. Preliminary studies, for example, have demonstrated a reduction of active rosettes in patients with malignancy and autoimmune disease before other parameters of ceil-mediated immunity were diminished. There are several other types of rosettes that have been distinguished, including giant and autorosettes.‘.” I”’ These will not be further discussed herein, but they illustrate, nevertheless, the critical nature of rosette formation and its ability in recognizing T-cell subpopulations. Most recently, selective growth and clonal proliferation of human T lymphocytes has been achieved using a single-phase semisolid methylcellulose medium in the presence of plant lectins (Fig. 3).“15m”‘i These techniques now permit direct visualization and cloning of individual T cells. it is noteworthy that the usage of T-lymphocyte growth with selective cloning now opens the possibility of study of both induction

Photomicrograph of a human T-cell clone in semisolid phase. Growth and characteristics Fig. 3. critical in our understanding of the clonal expansion of T cells. Hemotoxylin and eosin. x 150

of these colonies will be

LYMPHOCYTE

SUBPOPULATIONS

and amplification of T-cell function in vitro as well as the possibility of antigen recognition. Human T cells also have surface receptors for both the C-reactive protein and for measles virus.13* Additionally, human T cells can be identified by their reaction with rabbit anti-human thymocyte serum (RAHTS).‘3”.‘“o This antiserum is raised by the injection of rabbits with suspensions of human thymus and can be rendered T-cell specific by extensive absorption with B cells and liver to remove non-T-cell and general species determinants. As expected, RAHTS is capable of lysing T cells in the presence of complement and inhibits both mixed lymphocyte reactions and E rosette formation.140 Interestingly, in the absence of complement, RAHTS is itself mitogenic for T cells. Previous investigations have documented the cross-reactivity of anti-mouse thymocyte serum and mouse brain and have demonstrated the ability to produce xenoantisera to thymocytes by immunization of rabbits with mouse, rat, or dog brain. The production of anti-human thymocyte serum by immunization of rabbits with human brain, however, has been equivocal. Kongshavn et al. were unable to produce significant lymphocytotoxic antibody by immunization of rabbits with human frontal lobe.14’ Similarly, Rodt et al. were unable to absorb the activity of RAHTS with human brain.‘42 In contrast, others have reported succesful production of anti-brain serum cross-reactive with human T cells, using both adult and fetal brain, and Opelz et al. demonstrated that such antisera abrogated the ability of human lymphocytes to respond to, but not the ability to stimulate, a mixed lymphocyte reaction.‘43 The failure of some groups to produce such antisera from human brain is unclear but may be related to the viability of the brain and the interval between the patient’s death and its remova1.‘3g Rabbit anti-human brain serum (RAHBS) shares a number of characteristics with antithymocyte serum. In the presence of complement, it specifically lyses T cells. Thus it prevents formation of E rosettes and prevents stimulation by concanavalin A, phytohemagglutinin-P, and allogeneic cells. Other workers have reported that anti-human brain serum, prepared by immunization of rabbits with fetal brain, is reactive only for specific peripheral blood lymphocyte cell populations.‘44 This latter

255

cell population represents approximately 23% of circulating peripheral blood lymphocytes and is not further defined on the basis of either formation of E rosettes or active rosettes. It is of interest that the lymphotoxic antibodies found in murine and human SLE cross react with brain and that similar antibodies are found in patients with multiple sclerosis.‘3”*‘4”.‘46 There is also evidence that T cells possess membrane immunoglobulin. Unfortunately, the quantity of Ig present is too small to be detected by direct immunofluorescence.‘47-‘4g Using more sophisticated procedures, including electron microscopy of labeled antibody to immunoglobulins (e.g., turnip yellow virus), iodinelabeled antibody, and radiolabeled antisera, detectable membrane immunoglobulin can be demonstrated.‘4R A somewhat different approach can be used to quantitate the effect of antisera to Ig on alterations of antigen binding on select functions of T cells. Antisera to Ig inhibit antigen binding, antigen-specific helper function, production of macrophage inhibition factor, graft-vs-host disease, and antigen-specific “hot antigen suicide.“‘““*‘“” Such antisera do not inhibit the mixed lymphocyte reaction. However, blocking of these specific functions does not necessarily imply that surface membrane Ig is the antigen being blocked. This point has been dramatically illustrated by the blocking by the IgG fraction but not by F(ab) monomers of antigen-specific proliferation of T ceils in vitro.‘4g-‘“2 Analysis of rat and pig thymocyte plasma membrane protein reveals approximately 0.2%2% immunoglobulin.‘“2~‘s3 Needless to say, the issue of the nature of this membrane immunoglobulin continues to be a challenging one. It is therefore quite apparent from these arguments that cell-mediated responses are the domain of T cells. In this respect it is particularly interesting to comment on the elevated risk of allergy and autoimmune phenomena in patients with selective agammaglobulinemia and other congenital immune deficiency states.g6 The presence of rheumatoid arthritis, either without rheumatoid factor or subcutaneous nodules, in such patients is a wellclinical known observation.‘“4,‘5F, The mechanism for this association will be discussed and amplified below but suggests that in the

256

ROBBINS

absence of one of the major components of the dual system of immunity the other component becomes hypersensitive and is at least partially unregulable.‘54*156

Table 5. Characteristics

Possess

Fc and C’3 receptors

Prowde primary defense agamst high-grade encapsulated pathogens Possess

OF B CELLS

wrus

Respond wth

blast transformatmn

column

to the maogens I~popoly-

saccharlde. purified protein derwatwe and pokeweed

present on their cell surfaces that correlates with the antibody class produced. Specifically, B cells provide primary defense against highgrade encapsulated bacterial pathogens. ‘.‘.‘* It therefore is not surprising that patients with multiple myeloma or agammaglobulinemia suffer from recurrent pneumococcal infections. Additionally, B cells are critical for the detoxilication of protein polysaccharides and toxins (Tab,eQ4.“,‘.“’ B-cell function in humans can be rapidly evaluated by quantitating the serum level of three major immunoglobulin classes, IgM, IgG, and IgA. Moreover, the titer of antibody to several ubiquitous antigens as well as the isoagglutinins, antistreptolysin 0 (ASLO), and antiviral antibodies provides significant information on previous antibody formation. B cells, in a manner similar to T cells, can be stimulated to blast transformation with specific mitogenic agents, particularly lipopolysaccharide (LPS) and purified protein derivative (PPD).“” In contrast to T cells, however, B cells do not form spontaneous E rosettes with sheep blood cells. They do, however, form rosettes with erythrocytes coated with antibody and complement (EAC, erythrocyte anti--- -~--

-

EAC ROSETTE

IrlM

T-CELL

and polysaccharides

receptor for Epstein-Barr

Can be removed with antummunoglobulln

described above, B cells in chickens originate from the bursa of Fabricius. Mammals, however, do not have this organ, and a bursa equivalent has not yet been elucidated. Moreover, unlike the chicken, there may not be an exclusive source of B cells for mammals. Several groups have nonetheless proposed, based upon comparative considerations, that the bursa equivalent exists in mammalian gutassociated lymphoid tissues (the GALT hypothesis). However, removal of the gut in fetal lambs does not interfere with B cell maturation.4,‘“7-‘5g Alternatively, bone marrow, also a candidate, does not appear to be a bursa equivalent because its obliteration with ““Sr does not significantly alter B-cell ontogeny.4*‘8.‘5g There have been other candidates for the bursa, including fetal liver and spleen, all of which may be bursalike contributors.‘8.‘60.‘6’ In the balance, however, it appears that multiple factors/organs may be involved. It is noteworthy in this regard that transplantation of fetal liver into humans with severe combined immunodeficiency (i.e., without T or B lymphocytes) may result in the appearance of both mature B cells and immunoglobulins.‘62 The major function of B cells is the secretion of immunoglobulins and the differentiation into B cells plasma cells.‘63-‘“7 Indeed, individual have a receptor-specific immunoglobulin class ~ ----1

of S Cells

Secrete antibodies

As

E-ROSETTE

GERSHWIN

Contam surface lmmunoglobulms

Detoxify proteins. polypeptldes.

CHARACTERISTICS

AND

COMPLEMENT

EAC CELL

SW’S E-CELL

196 ANTI OX-REC

SENSITIZED 0X-M

I’ 8 LYMPHOCYTE ; OR ACTIVATED I T-CELL

EAC CELLS

SENSITIZED OX-RBC

EAC %ETTE

Fc-ROSETTE

Fig. 4. Schematic representation in the formation of human E. EAC, and Fc rosettes. Note specific target cell responsible for formation of rosettes in each of the schema.

LYMPHOCYTE

SUBPOPULATIONS

body-complement rosettes).‘64 Such EAC rosettes depend upon the presence of receptors for C’3 on the surface of B cells (Fig. 4). It should be emphasized, however, that monocytes also have a receptor for C’3 and thus form EAC rosettes. In contrast, however, B cells form rosettes with either mouse or monkey red cells and have surface receptors for Epstein-Barr virus (Table 5). Of particular importance to the identification of B cells is the presence of surface immunoglobulin. During differentiation of B cells, p, y, and (Y constant-region genes are sequentially transcribed during clonal expansion.‘6* Indeed, as noted above, the expression of all classes of B cells in neonatal mice can be chronologically suppressed by repetitive injections with antiP. ‘g,20The switch sequence appears to be, in order, IgM to IgG to IgA. However, there has been considerable recent attention to the appearance of IgD and IgM concurrently on the surface of fetal and cord lymphocytes.‘6g*‘70 This has suggested that expression of the 6 gene is an early event and that it may be critical in the ontogeny of B lymphocytes.“’ In any case, the presence of surface immunoglobulin is the most commonly used characteristic to directly identify B lymphocytes. The method commonly employed has generally been direct immunofluorescence with a fluorescein-conjugated heavy chain-specific antiimmunoglobulin (Fig. 5). “2 Unfortunately, accurate identifications of intrinsic surface membrane immunoglobulin is subject to a number of serious experimental artifacts. In particular, both B cells and monocytes have Fc receptors on their surface, and Fc receptors will bind protein aggregates present in the antisera (Fig. 4).“’ Ironically, commercial antisera are often packaged in lyophilized form, and the final product may therefore have a considerable amount of aggregation. This will cause fluorescence of Fc receptors and will be optically indistinguishable from the staining of surface immunoglobulin; thus artificially high values may be experimentally obtained. 174This problem has been under intense investigation because it appears the majority of studies that quantitated and reported the number of peripheral blood B cells may haveoverestimated them. Fortunately, this experimental error can be reduced by ultracen-

257

DIRECT

TEST

llll111llll ULTRA

INDIRECT

VIOLET LIGHT

TEST

_,UNLABELED

h

’

L

ANTIBODY

a 3

tlltllllltl ULTRA

VIOLET LlGHT

Fig. 5. lmmunofluorescent staining of tissue sections using either direct or indirect test. This procedure can be modified for single-cell suspensions and illustrates the versatility of this method.

trifugation of antisera prior to use to sediment and thereby remove aggregates. Similarly, one may use F(AB), antisera. Additionally, it is critical to ascertain that the surface immunoglobulin is indeed synthesized by the cell and does not represent cytophilic antibody. 175In SLE, for example, antithymocyte antibodies coat T cells and may fluoresce when mixed with a fluorescein isothiocyanate (FITC) anti-Ig. This continues to be a problem, but it may be minimized by washing cells prior to staining to permit elution of cytophilic antibody. Study of surface Ig, following its resynthesis after previous enzymatic treatment, distinguishes endogenous immunoglobulin from immunoglobulin passively acquired from sera and sticking because of Fc receptors (Table 6). The relative and absolute number of peripheral blood B cells has not yet been stanTable 6. Detection

of Membrane-Bound

Fluorescent microscopy

using

lmmunoglobulin

FITCantisera or FITC

FIAB)z antisera RhodamIne

conjugation

Enzyme conjugation F&tin

conjugation

Autoradiography Radioimmunoassay -

of antisera Iperoxidase) of antisera

258

dardized. Moreover, a significant percentage of B cells have shed their immunoglobulin and will be undetected by simple direct immunofluorescence. Nonetheless, it is believed that the relative number of peripheral blood surface immunoglobulin-positive cells is l%-~ 15%.““-“” The majority of these cells bear both IgM and IgD. The total number of IgGand IgA-bearing cells is less than 2% 5%. The number of complement receptor-bearing lymphocytes is similarly in doubt because of a heterogeneous population. It is known, however, that the two major complement receptors include C’4 or the C’3c region of C’3b, i.e., the immune adherence receptor. Alternatively, the C’3d receptor is specific for the C’3d region of C’3b.jZ Both receptors are generally present on individual cells but may be independently expressed, particularly in lymphoproliferative disease, I;;. lib As noted, it is clear that the predominant immunoglobulin on B cells of normal individuals includes both IgD and IgM.17” Although this observation initially appeared somewhat surprising in view of the low levels of serum IgD, it is now proposed that IgD may be critical in the clonal development of B cells and in the ontogenetic triggering of B-cell maturation.‘xu~lx’ In contrast, the mechanism for development of IgE and its ontogenetic expression has not yet been clarified.‘X”p’h’ However, as with IgA, its production may be under thymic influence and its level may be increased by treatment with radiation and antilymphocyte sera.“‘~“’ More recently, it has been demonstrated that there are naturally occurring antisera in multiparous women as well as in multiple transfused patients that recognize B-cell alloantigens. Ix5These sera also contain antisera for HLA-A, -B, and -C determinants, but these can be removed by absorption with pooled outdated human platelets.“” (Platelets, although possessing HLA-A, -B, and -C markers, do not express B-cell alloantigens). It is now possible to isolate specific antigens responsible for these cell markers and accordingly to produce B-cell alloantisera in rabbits.‘“” Recent studies of large numbers of such sera have led to the demonstration that such B-cell markers may be analogous to the Ia region of mice.‘*” If such proves to be the case, the classification of B-cell

ROBBINS

AND GERSHWIN

alloantigens will be particularly critical in understanding the pathogenesis of immunologic disease. Indeed, dramatic associations of multiple sclerosis, gluten enteropathy, asthma, and childhood leukemia have recently been demonstrated with respect to presence or absence of particular specific B-cell antigens. Ixi CHARACTERISTICS

OF NULL CELLS

In both mice and humans,

subpopulations of lymphocytes lacking surface marker characteristics of both T and B cells have been demonstrated. Briefly, such cells form neither E nor EAC rosettes and lack surface immunoglobulin. They can be further distinguished from monocytes by their inability to phagocytize latex. ‘x7-‘xo However, null cells, as they are thus referred to, possess Fc receptors and function as efficient effector cells for antibodydependent lymphocyte-mediated cytotoxicity (ADLMC).‘“” In ADLMC they have been referred to as K cells. However, it appears highly likely that null cells represent a heterogeneous population. It is intriguing that the percentage of null cells in human peripheral blood is highest for patients with autoimmune disease, e.g., SLE.‘“‘.‘“‘,‘!“,‘!‘” There are a number of ways, as discussed below, to selectively enrich null cells.‘!“‘+‘“” Moreover, there is some evidence suggesting that null cells are destined to differentiate into B cells. Thus they may represent either B cells that have shed their immunoglobulin or, in contrast, early uncommitted cells (Table 7). SEPARATION

OF HUMAN

LYMPHOCYTE

SU BPOPULATIONS

There are a large number of techniques currently being used for the isolation, fractionation, and characterization of human and murine lymphocyte subpopulations.‘g”+“X Unfortunately, many of these are based on methods that Table 7. Characteristics Far1to form spontaneoos

of Human Null Cells

rosette wafh sheep erythrocytes

Lack surface lmmunoglobulw Effectwe I” wtro as effector component dependent

cell-medlated

I” armbody-

cytotoxnty

Possess Fc receptors Nonadherent Nonphagocytlc Morphologically

lndsnngulshable

Probably a hetarogenous transIttonal

6 cell

from T or E cells

population:

may represent a

LYMPHOCYTE

SUBPOPULATIONS

259

have not been standardized, and all suffer from the hazards of nonspecific cell loss and alterations of membrane properties. All have in common the ultimate desire to purify a population of immunocompetent cells with specific surface membrane receptor and functional characteristics. Separation procedures have been considerably more successful in mice because of the large numbers of inbred murine lines available. Indeed, inbred mice have permitted the identification of several T- and B-cell alloantigens; such alloantigens have helped enormously in the characterization of individual lymphoid subpopulations.2”-206 Humans, in contrast, despite some degree of linkage disequilibrium, remain an outbred population, and only a few such genetic markers have been described with relevance to lymphoid subpopulations.“,‘x.‘“d Separation procedures can be subdivided into those based on physical properties of cells and those based on surface markers (Table 8). The most commonly used method to separate peripheral blood lymphocytes from granulocytes and red cells of whole blood is based on the density gradient centrifugation of Boyumlgx (Table 8). Briefly, whole blood is diluted at least 1:1 with either isotonic saline or medium and layered over a Ficoll-Hypaque mixture, density 1.077. The blood is centrifuged, permitting the separation into two fractions: a white layer containing lymphocytes, monocytes, and platelets at the interface region and a lower fraction containing erythrocytes and polymorphonuclear leukocytes.‘“R The lymphocyteenriched fraction is readily removed with a Pasteur pipette and can be washed to remove platelets; a yield of more that 90% lymphocytes Table 8. Methods for Separation

of Human

B and T Lymphocytes Utllizmg

-

surface markers

Cyrox~c antwera (anti-lg. anti-B. anti-T) Column fractionation Rosette sed,menratlon Absorbent

(anti-lg.

monolayers

Fluorescence

activated cell sortmg (anti-lg. anti-B. anti-T)

Utlllzlng general

propertm

Denstty gradtent (d,fferentlal dnxntinuous Sedimentatmn Adherence

anti-FlabL]

(E. EA. EAC)

Flcoll-Hypaque

BSAl at lg

columns (nylon wool)

Electrophoresis

and

can be obtained. The monocytes in this fraction are removed with considerably more difficulty. In particular, they can be depleted by taking advantage of their ability to phagocytize carbamyl iron.2o4 Such cells become heavier and can be drawn to the bottom of test tubes either with a magnet or by recentrifugation over FicollHypaque. Unfortunately, the majority of monocyte-depletion techniques result in the dramatic loss of more than 30%-40% cellszO” Hence attempts to selectively enrich in lymphocytes following Ficoll-Hypaque centrifugation may result in a considerable cell bias. In several disease states, such as SLE, the cell loss following centrifugation may be considerably enhanced over the cell loss resulting from the blood of normal volunteers.‘“4.‘gl Studies comparing disease states with normals therefore may be fraught with error. After obtaining an enriched population of lymphocytes from whole blood, other density centrifugation procedures can be employed to isolate subpopulations of cells. These include both continuous and discontinuous bovine serum albumin (BSA) and Ficoll ‘R’.‘9’,204-2”X Continuous BSA gra_ gradients. dients have hitherto been the best and most versatile system. Cell populations separated on BSA gradients appear heterogeneous based on several criteria including homing studies, life span, surface markers, and function in vitro and in vivo.‘“1.2”“-20” Briefly, a 35% BSA gradient in Tris buffer is prepared and adjusted to 360 mOsm with NaCI. Thereafter, either a continuous or discontinuous gradient is established from 19% to 35%. Experimental cells are suspended in 17% albumin, applied to the top of the gradient, and centrifuged at 500 g. Individual fractions can then be removed and studied. Using this procedure, the selective deficiency of B cells in agammaglobulinemia can be demonstrated (Fig. 6). A similar but much simpler principle is the fractionation of cells based upon the unit gravity sedimentation technique.*O’j Cells are suspended in a variety of media and allowed to sediment unhindered at 1 g. Generally, the media employed is phosphate-buffered saline containing human or bovine serum albumin. The function of the protein is to prevent convection and mixing of adjacent layers during the loading and emptying of the sedimentation chamber. Using

260

ROBBINS

CENTRIFUGED 9009 r45’0,

IO0 c

1

HIGH RATE OF DNA SYNTHESIS DECREASED MITOGENIC RESPONSIVENESS FORM COLONIES WHEN CULTURED FAIL TO STAIN WITH MYELOPEROXIOASE.

IMMATURE LARGE PROGENITOR CELLS T-CELLS

!

7

,kCELLS

8

AND GERSHWIN

VIGOROUSLY STIMULATED BY MITOGENS ANTIGENS, ALLOGENEK CELLS, FORM E-ROSETTES; TAKE UP ANTIGEN; SECRETE CELLULAR MEDIATOR: i MITOGENIC FACTOR

Fig. 6. Separation of T and B cells using discontinuous BSA gradient. This procedure allows for the separation of lymphocytes according to size and density. Note that functional studies confirm the enrichment procedures.

STAIN POSITIVE FOR Sk, AND Fc FORM EAC ROSETTES DO NOT PROLIFERATE TO ’ ANTIGENIC STIMULI

I

9 I

this procedure, cells are readily separated on the basis of their sedimentation rates. The settling pattern (plot of cell number versus settling rates) is strikingly similar for normal In mice this procedure allows for volunteers.‘o’ the enrichment of null cells, T helper and suppressor cells, and B cellszo6 Another feature that can be used to distinguish T from B cells is the individual expression of surface electrical characteristics.213.2’4 All mammalian lymphocytes contain certain surface charges, and cells can be separated according to their different anodic mobilities. By using analytic preparative methods, B cells have been demonstrated to possess a lower electrophoretic mobility than T cells.2’3.“‘4 There are several individual variations within T- and B-cell populations suggesting that subpopulations of cells may be recognized by this property. Finally, major differences in the net charge of lymphocytes in several diseases, including rheumatoid arthritis, have been demonstrated,‘“V2K3.21’ Rosette techniques can also be used to selectively enrich T or B cells.13” Briefly, E rosettes are formed as above and then recentrifuged on a Ficoll-Hypaque gradient. Cells that form rosettes (T cells) sediment to the bottom layer, whereas B cells and monocytes remain at the interface. Thus selective enrichment of both Erosette-positive and -negative populations can be attained. These procedures can be enhanced by stabilizing rosettes in dextran buffers and by other means but are optimally used primarily for the gross separation of T from B cells.‘3g They are of little value in the separation of

subpopulations or in the enrichment of antigenspecific cell populations. The functional and critical importance of antigen-binding cells and their use in cell enrichment techniques was recognized by Andersson and co-workers.““.*” They observed that passing a population of previously immunized lymphocytes over an antigen-coated glass bead column led to the selective depletion of antigenreactive cells (Fig. 7). Both antibody-producing cells and their precursors could be removed in this manner.“‘,“” Finally, through the use of

IMMUNIZE0

WITH HSA

SUSPENSION

IMMUNIZED WITH ESA

OF POOLED SPLEEN CELLS I

I

i ANTIGEN

ON COLUMM

HSA

ESA ‘,I,_ ,1

ILr

1

RErAlNED CELLS

?LSifSD

1I PASSED CEI.LS

“%zED

Fig. 7. Affinity chromatography for the isolation of antigen-specific lymphocytes. This procedure can be used in a variety of animals. It has as its major handicap a high cell loss. It is, however, an important procedure for the study of antigen-specific cells.

LYMPHOCYTE

261

SUBPOPULATIONS

enzymatic digestion the antigen-specific populations binding the beads could be eluted and recovered.200~21’~2LZ The specific affinity of lymphocytes for coated glass or plastic bead columns can be easily adapted for a variety of antigen-binding receptors. It is now possible to fractionate cells according to large numbers of surface markers, including receptors for activated complement components, Ig molecules, Fc receptors, and surface antigens.2”,2’2 Generally, beads composed of either glass, plastic, cross-linked dextrans, agarose, or acrylamide have been used; such coated materials thus can potentially possess the ability to bind a subfraction of cells. These columns are extremely efficient, and more than 90% of surface Ig-positive cells can be removed and recovered. Unfortunately, there may be a great cell loss. Moreover, such columns are relatively poor at removing antigen-binding T cells. Several other methods have been studied. One such method involves the fractionation of peripheral blood lymphocytes on snail (Helix pomatia) hemagglutinin coupled to sepharose beads.lgg It has been demonstrated that passage of lymphocytes previously treated with neuraminidase over snail hemagglutinin coupled to sepharose separates cells into two populations; helix-positive (i.e., population that binds to the coated sepharose) and -negative populations; this allows further fractionation. This method has permitted the highly reproducible enrichment of EAC- and E-rosette-forming popuiations.lYg Many laboratories employ several of these procedures in attempts at selective isolation of subpopulations. Moreover, the field is an extremely active one, and newer procedures continue to be developed. In particular, separation and characterization of cells based upon density of surface membrane immunoglobulin, using a fluorescence-activated cell sorter, is rapidly becoming one of the most unique and versatile tools in immuno10gy.“L6-2’8 The fluorescence-activated cell sorter was designed to separate lymphocytes on the basis of fluorescence and size by confining the cells of interest to the center of a liquid stream, thereby forcing them to pass individually through the focused beam of a high-power laser. Each lymphocyte is identified and characterized on the basis of the amplitude of the signals, which

correspond to the intensity of light scattered by the cell while it is in the laser beam. Such signals can be processed based upon previously selected criteria and identified as to whether or not they should be separated from other cells within the stream. This separation is performed by forcing the liquid stream through a small ultrasonically excited nozzle, causing the stream to form into equal-volume drops. The cells of interest are then electrically charged and deflected by passage through an electric field. A profile of the cells under study can be quantitated and cells of interest removed for further study.2’fi-21R REGULATION

OF THE IMMUNE

RESPONSE

The immune responses controlled by T cells are at least from an ontogenetic viewpoint independent of B cells.4~“~L”4~2’9-‘“3The concept, however, does not imply that their physiologic roles are mutually exclusive. In 1966, during studies of the functions of T and B cells in the production in vivo of antibody to SRBC, it became clear that a profound synergism existed between thymocytes and bone marrow cells.26~‘20 Insignificant titers of antibody were produced in either T- or Bcell-deprived animals. p6,27This critical observation introduced the concept of positive interactions in immunity and led to the doctrines and formulations proposing that antibody to thymicdependent antigens depends on the interaction between antigen, monocytes, T helper cells, and B cells.“~“fi~‘“4~2”“~224 Moreover, similar studies have demonstrated that two different subpopulations of T cells positively cooperate in the induction of graft-versus-host disease and skin allograft rejection.233 Until recently, the vast majority of efforts directed at unraveling the events leading to production of the immune response against SRBC, autoantigens, and even infectious organisms have been directed solely at the positive factors involved in regulation, namely, the factors responsible for augmentation. It has been postulated that T cells, under the influence of antigen, become activated and release a special immunoglobulin molecule known as IgT. 226--228The structure and function of IgT remains speculative but may be analogous to the immune response gene. Furthermore, the function of IgT may depend heavily on the

262

genetic relationship between T cells and antigen recognition/responsiveness. It is also well known that immunity represents a double-edged sword. There are numerous examples of the detrimental influence of the immune system.‘“’ In many of these detrimental situations, the innocuous influence upon the host is due to an immunologic hyperresponsiveness either in the form of allergy or as autoimmune phenomena.‘“4 Therefore attempts at understanding the development and expression of the immune response are critical if immunity is to be manipulated and adjusted to prevent harmful effects. It is through our study of autoimmunity and responsiveness to both self- and foreign antigens that the theater of immune regulation may be introduced. Immune regulation is inherent in the concept of the dual-component pathway of the immune system responsible for the differentiation of antigen reactor precursor cells and the subsequent development of either cell-mediated or humoral immunity.‘““-‘“’ As noted earlier, the thymus gland and the bursa equivalent produce stem cells that differentiate to form immunocompetent antigen-reactive cells. Production of antibody is directly related to the development of precursors of antibody-producing cells (B cells), which recognize antigen by surface immunoglobulins specific for that antigen. Under this influence, B cells differentiate into plasma cells, proliferate, and secrete specific antibody.‘.“,‘“+ Similarly, a select population of B cells does not produce antibody but nevertheless differentiates and survives for long periods of time as memory ce]]s.“, 163--1RR These latter cells are capable of response following secondary challenge or exposure to antigen and therefore produce the anamnestic response. In contrast to humoral immune responses, cell-mediated immunity is directly under the influence of thymic-derived T cells. The mechanism by which T cells recognize antigens and produce positive signals, in contrast to the above information on B cells, is unclear. It is apparent, however, that under the influence of antigens T cells undergo repeated mitosis and differentiation into cells responsible for cellmediated immunity. Such stimulated T cells also produce several active soluble mediators, including lymphotoxins, skin reactor factors, in-

ROBBINS

AND GERSHWIN

terferon, and macrophage inhibition factor; each plays a significant role in delayed hypersensitivity, response to infectious disease, and activation of macrophages. Similarly, a subpopulation of T cells may likewise develop into long-lived memory cells and be responsible for development of anamnestic responses.“‘“-‘L”’ Additionally, under appropriate circumstances both stimulator T and B cells are capable of becoming tolerant to select antigens.“,“” Under this influence, tolerance or immune paralysis to either cell-mediated or humoral immunity is possible. )-i.~L;16.9:li It is also important to discuss the existence of a third cell population involved in positive regulation, the macrophage. Indeed, the macrophage appears important for the presentation of antigen.‘“+“” As such, development of the primary immune response and initiation of both cell-mediated and humoral immunity depends on the activity of monocytes.‘L4’~‘4’ The precise role of monocytes in both positive and negative immune manipulation is under intense investigation; however, it is apparent that they function in a large variety of ways and that there may exist subpopulations of these cells. Reviews of monocyte development and function have presented been elsewhere.~‘L-“’ The positive mechanism by which T cells result in the development of the humoral response by B cells to complex antigens has been demonstrated for hundreds of antigens.““-“’ For example, B cells both in vivo and in vitro are able to make significant antibody responses to the thymic-dependent antigen SRBC only under the influence of T cells.“)” This positive cooperative interaction appears to be regulated by surface receptors coded by genes of the major histocompatibility comcertain soluble mediators, plex.2.‘x Additionally, perhaps products of the I region of the H-2 complex in mice, are critical for mediating this cooperative interaction between T and B cells.~“!‘.““” Similarly, although precise surface receptors on T cells have not been identified, an immunoglobulinlike material known as IgT (see above), produced by T cells after interaction with antigen, may be important for presentation of B cells under the influence of monocytes. There have also been several demonstrations that subpopulations of T cells exist, distin-

LYMPHOCYTE

SUBPOPULATIONS

guishable on the basis of having different surface characteristics and physical properties; these have been best studied by comparing properties of murine T cells derived from thymus, spleen, and thoracic duct.‘50-‘56 Some such physical properties have been discussed above and include separation and classification of cells based on electrophoretic charge, and mitogen responsiveness. In density, particular, murine T cells can be further subdivided on the basis of unique mouse surface including 0, TL, and Ly receptors, antigens."53-255 Cells can be further characterized on the basis of cortisone and reradiation sensitivity. w These characteristics veal that murine T cells are an extremely heterogeneous population and can be readily isolated to characterize select discrete subpopuThe biologic relevance of this lations. heterogeneity is critical. It is further apparent that many of these differences are due to distinct stages in T-cell development.2”7 Based on surface receptors, these distinct populations may have specific immunologic properties, including, for example, positive (helper) or negative (suppressive) actions. In mice, these two contrasting features can be separated on the basis of a unique set of antigens known as LY.2n3 Thus data is now accumulating suggesting that the functions of T cells may be carried by different sublines of T cells. These different subpopulations may be specifically detected and isolated within the thymus as well as peripheral lymph node tissues. From a functional point of view it is accurate to state that study of graftversus-host disease, development of cytotoxic lymphocytes, and immunoglobulin production have all demonstrated that T cells can function both in a positive and a negative regulatory capacity to alter immune response.““7-‘77 In contrast to the development of positive immune responses, the influence of negative regulatory mechanisms (those that turn off or prevent development of immune responses) have been poorly investigated. Until recently, the entire subject of negative regulatory mechanisms was not even considered. Nonetheless, over the past several years a number of interesting experiments have led to the concepts of active suppression of immunocompetent cell populations by thymic-derived cells. Such active

263

regulation by suppressor T cells can be readily distinguished from the influence of specific antibody or antigen-antibody complexes in regulatSeveral studies of ing immune responses. 25H--260 negative feedback suggest that this suppression may play a critical role in autoimmune disease. “* In humoral immunity, suppression has been most easily demonstrated for antigens not requiring thymic helper ce11s.246-267 The antibody response to pneumococcal polysaccharide (SSS III) is enhanced by the administration of antithymocyte serum; this enhancement is reduced by the subsequent administration of 274 Ironically, this observation thymocytes. seemed to contradict previous concepts on the use of antilymphocyte serum as an immunosuppressive agent. However, these experiments were confirmed using another antigen, polyinosinic-polycytidylic acid (poly I . poly C). 278 These studies taken together strongly suggested the existence of thymic regulation by suppression of the antibody response to several antigens requiring little thymic helper function. suppression has also been In addition, demonstrated for antigens requiring thymic helper cells for a maximum antibody response."6,265.271 Similarly, the homocytotrophic (IgE) antibody response to dinitrophenyl-ascaris may be regulated by antigen-specific suppression.“72 In cell-mediated systems, suppression of the induction of graftversus-host disease by thymocytes and spleen cells has been demonstrated.“A’ The limited information to date suggests that more than one kind of thymic suppression may exist, including nonspecific and specific suppression of antibody responses and suppression of cellular immunologic phenomena. Further study may disclose more subdivisions.*’ The mechanism of suppression is poorly understood. Preliminary results suggest that in the antibody systems thymic suppression may act by inhibiting B-cell proliferation.*‘j3 A similar suppression of T-cell proliferation would be plausible for the cellular system, including graft-versus-host disease. In the normal host, however, there probably exists a balance between suppressor function and proliferative functions.276.277,27g-*86 Chronic allogeneic disease may be an example of relative reduction in suppressor function secondary to a

264

pronounced enhancement of proliferative function.‘54 This negative influence of T-cell populations on the immune response may be either antigen specific or nonspecific. An example of nonspecific suppression of immunity includes the phenomena of antigenic competition, mediated by thymic-derived cells previously stimulated by the antigen, inducing competition.86,L”4 This can be illustrated in the acute production of anergy in guinea pigs. For example, guinea pigs allergic to a variety of antigens can be rendered anergic to all of these antigens by the injection of a large quantity of one antigen in soluble form.X6.263 Although this anergy is short lived, it can be prolonged by repetitive injections of the antigen but not overcome by injecting sensitized lymphocyte cells into anergic test animals.86*“63 Nevertheless, lymphoid cells from anergic guinea pigs are capable of transferring delayed hypersensitivity to normal guinea pigs. The etiology of this form of antigenic competition appears to be secondary to a number of local factors, including soluble mediators produced by suppressor T ce11s.273-277*27”-284 Other mechanisms, including influence of the reticuloendothelial system, competition among determinants on complex antigens, and local competition with lymphoid tissues, are also critical. A number of experiments directed at explaining antigenic competition have heavily incriminated soluble mediator(s).‘7”P”77.279--284 A variety of protein substances released by antigen-specific stimulated T cells have been described, all of which appear to reduce the immune response to other related antigens.‘72 Although the precise chemical nature of the mediator(s) has not been determined, it is distinguishable from soluble materials produced by helper T cells in the development of positive immune responses. As will be shown below in regard to studies of concanavalin A on the immune response, a number of biologic properties of this material are now known, and preliminary studies have suggested major clinical applications 283,284 The identification of specific populations capable of negative regulation of the immune response led to attempts to selectively isolate and characterize this population. In particular, suppression has now been demonstrated in a large number of systems both in vitro and in

ROBBINS

AND GERSHWIN

vivo. For example, thymocytes can suppress both cell-mediated and humoral immune responses when injected into intact syngeneic recipients.“63 Moreover, the antibody responses to thymic-independent antigens can be enhanced by either previous injection of antithymocyte sera or by thymectomy.x”~‘“4~2”” Similarly, it has been demonstrated that the spleen is a rich source of suppressor cells, and this splenic enrichment is particularly high in very young mice. zx6 Hence the lack of significant maturation, i.e., the failure to produce significant humoral immune responses in young mice, may be due to the enrichment of suppressor cells in these animals. The activity of suppressor T cells on the expression of autoimmunity has received considerable attention, particularly in New Zealand mice. New Zealand black (NZB) mice develop a significant Coombs’ positive hemolytic anemia with age. New Zealand white (NZW) mice, in contrast, develop a low titer of antinuclear antibodies and mild glomerulonephritis in older animals.“” The mating NZB x NZW produces an F, hybrid that develops a disease similar to human systemic lupus erythematosus.‘X7,““X Such mice spontaneously develop antibodies to nucleic acids (DNA), immune complex nephritis, and nephrotic syndrome. Furthermore, both NZB and NZB/W mice are well characterized as being hyperresponsive early in life, compared to other strains of mice, to a variety of antigens.Yxx,2”!’ Their immunologic system appears to mature at a more rapid rate. These observations led to the suggestion that autoimmunity in these mice, and perhaps their related clinical human analogues, stems from a premature loss of suppressor cells.‘go~‘9’ Following neonatal thymectomy, autoimmunity is accelerated. This can be corrected by syngeneic thymus grafts from 2-wk- but not IO-wk-old mice, suggesting an age-associated loss of suppressor cells.2yo-“g2 Further evidence supporting this concept includes the observation that young but not old New Zealand mice show antithymocyte serum-induced enhancement of the antibody response to poly I - poly C.“” In addition, the antibody response to SSS III increases with age in New Zealand mice, unlike control mice, and can be suppressed with young thymocytes.27x

LYMPHOCYTE

SUBPOPULATIONS

Finally, spleen cells from 6-mo-old New Zealand mice induced a more vigorous graft-versushost response than spleen cells from 6-wk-old New Zealand mice.26’ The graft-versus-host response induced by spleen cells from 6-mo-old New Zealand mice can be suppressed by young spleen cells or thymocytes.26’ It is well known that it is extremely difficult to produce tolerance in New Zealand mice, again, perhaps, by virtue of their premature loss of suppressor population.278~2g3-2g5 Because New Zealand mice are highly inbred, this loss of suppressor cells lends itself to considerable immunotherapeutic manipulation. It has been demonstrated that repetitive treatment of NZB mice with young syngeneic thymocytes markedly diminishes the expression of autoimmune disease.2g2 The cell population responsible is both radiation and corticosteroid sensitive. Additionally, it has been demonstrated that weekly treatment of NZB/W mice with young syngeneic spleen cells likewise diminishes the expression of autoimmune disease. Obviously, the mechanism for reduction of this disease is critical if similar therapy can be considered in humans. Nonetheless, because humans are outbred, repetitive inoculation of allogeneic lymphoid populations may result in considerable damage to the host, including possible development of graft-versus-host phenomena, chronic allogeneic disease, and severe allergic phenomenon. Thus the mechanism by which suppression is mediated will be essential for any practical application. In this regard, it is particularly interesting in studies of suppression to note that T cells derived from mice previously injected with concanavalin A are capable of suppressing the immune response to SRBC both in vitro and in vivo.2g6,2g7Such observations have led to a number of intriguing experiments relating to stimulation of a specific suppressor cell population by con A. Indeed, although con A itself may under certain circumstances be suppressive, it was apparent that in these negative mechanisms this suppression was mediated by a soluble production produced by the interaction of con A and lymphocytes.2g8~2ggWith the availability of specific T-cell alloantisera, it was demonstrated that con A acts specifically on Ly2,3+ T cells.2g6-300Moreover, con A can produce supmaterial in the pressor absence of

265

macrophages, and the site of action of con A appears to be a relatively radioresistant population. Study of the products released by stimulation of con A revealed the mediator now known as soluble immune response substance (SIRS).30’ SIRS in vitro can suppress the response to both thymic-dependent antigens, e.g., SRBC, and thymic-independent antigens, e.g., DNPFico11.2s’-284Critically, this suppression is not mediated by any direct cytotoxicity, because the number of viable cells following exposure to SIRS remains relatively unchanged. SIRS appears different from soluble mediators produced by the interaction of helper T cells and antigen.257-263Stimulation of SIRS by con A appears, at least at present, to be relatively specific because this material is not produced by a number of other T-cell interactions, including mixed lymphocyte cultures and transformation by phytohemagglutinin.300-30’ The chemical properties of SIRS include resistance to DNase and RNase but destruction by trypsin. Electrophoretically, it migrates in the postalbumin region of the beta globulins. Additionally, it is stable at 56°C for 1 hr but is destroyed at 70” C within 10 min. Of critical importance is that SIRS has no strain specificity and does not appear to be significantly antigenic.301.302Thus supernatant fluid containing SIRS produced by the interaction of con A and lymphocytes can be used to regulate immune responses. Spleen cells from older New Zealand mice appear not to liberate SIRS upon contact with con A, further suggesting the concept of loss of suppressor cells in the pathogenesis of autoimmunity in New Zealand mice.‘54.2g5 The potential clinical use of SIRS has been seized upon by a number of laboratories. Of particular interest is the major and significant reduction of disease in New Zealand mice by repetitive weekly treatment in vivo with SIRS.303 If the report of successful prevention of autoimmunity by SIRS in mice can be demonstrated in humans, it would lend itself to immediate and major therapeutic manipulation 303-310 The role of suppressor cells as significant in the development of the immune response has been shown for systems other than autoimmunity. In particular, it has been demonstrated that suppressor T cells are involved in

266

expression of common variable hypogammaglobulinemia.85~86 In this disease, previously thought due entirely to absence of specific B cells, it has been shown that T cells from patients with hypogammaglobulinemia are capable of suppressing immunoglobulin production by B cells from normal patients. Although these patients are unable to produce significant quantities of immunoglobulins in vivo, it can be demonstrated that in vitro and in the presence of normal T cells they are capable of Ig production. Hence the development of common variable hypogammaglobulinemia may be a direct result of the presence of a suppressor cell population.x” To what extent this population represents a clone of cells present in all individuals, but removed with age in most, is unclear. However, this population now lends itself to consideration of therapeutic manipulation, and appropriate medication and/or antisera can be used in vivo to eliminate this population. This consideration is now being applied to a similar hypogammaglobulinemia disease in chickens.2” Finally, the significance of suppression of the immune response can be further emphasized in the incrimination of suppressor T cells in development of aplastic anemia.““.‘“’ It is well known that erythroid aplasia may be accompanied by a thymoma, and resection of the thymus often returns red cell production to normal. Although this influence of the thymoma on red cell production has been known for some time, its role or influence has largely been ignored. However, recently it has been demonstrated that T cells from patients with aplastic anemia are capable of suppressing normal bone marrow production in vitro.“” Such T cells appear to be functioning as a suppressor population. Thus the influence of this population may extend into the development of hematopoietic precursor cells and may incriminate a common embryologic pathway for both hematopoietic and immunopoietic cell populations. Although the above description of suppression has strongly implicated the existence of suppressor T cells, it is nonetheless becoming equally true that suppressor B cells also exist.‘” Furthermore, a negative role in immunity can be demonstrated for monocytes.‘“.‘“’ Thus the role of suppression in several facets of immunoregulation may be mediated by virtually all major classes of mononuclear elements.

ROBBINS

ABNORMALITIES

AND GERSHWIN

OF LYMPHOID

SUBPOPULATIONS

IN

DISEASE

Consideration of abnormalities of major lymphoid subpopulations should commence logically with congenital primary immunodeficiency syndromes (Table 9). In these diseases, two major patterns have been observed, absence or near absence of T or B cells in the circulation, and normal or elevated levels of B cells but decreased serum immunoglobulin and decreased plasma cells in peripheral tissue. Infantile sex-linked agammaglobulinemia is an example of absence of B cells as detected by surface immunoglobulins, Fc receptors, and distribution of cells on a continuous BSA gradient. ’ Interestingly, such patients also have a marked increase in lymphocytes reacting with anti Tcell alloantisera. Selective IgA deficiency represents the second type of pattern. In this Table 9. T and B Lymphocyte Status in Disease Lymphocyte S,N”S o,sease

T

Predominant Lymphocyte Pllen0type

B

E kxetre

1

+

,g+

C’3

Fc

-

7 a,, norma,

N N N

1

1

N

+

+

N

1 N

+

+ or

+

+ +

t N

+

t N

I t

+ + + +

N N

+

1

t

+ +

N

+

+

p,1

+

+ +

+ N

1

+ +

t

+ + +

:

1 N

+

+

+

alI deceased

1 N

+

1 +

+

LYMPHOCYTE

SUBPOPULATIONS

disease, cells bearing surface IgA are usually normal or perhaps elevated; nonetheless, there is an absence of sera and IgA secretory plasma cells.“7 Interestingly, IgA synthesis is defective in cultured lymphocytes from affected patients. However, this failure to produce IgA may be related to a suppressor population. Indeed, T cells from patients with selective IgA deficiency are capable of inhibiting IgA production by normal individuals, a phenomena similar to that discussed above for common variable hypogammaglobulinemia.x” In contrast to these two major patterns of congenital or primary immunodeficiencies, there are a number of acquired lymphoproliferative diseases characterized by a monoclonal proliferation of subpopulations of cells.‘““-J’” It is now apparent that a considerable number of lymphoproliferative disorders are capable of being recognized as monoclonal B-cell diseases.“‘:‘+J”. The best example is chronic lymphocytic leukemia, which appears to have a specific monoclonal immunoglobulin on its cell surface.““-““’ Individual patients have surface immunoglobulin of either K or A origin, further implying the monoclonal origin of this malignant population. Similarly, both allotype and idiotype studies further suggest this unique clonal origin. These characteristics of monoclonal cell populations can be demonstrated in chronic lymphocytic leukemia, cold agglutinin disease, lymphoblastic leukemia, hairy cell leukemia, and macroglobulinemia (Table 9). Thus these lymphoproliferative diseases appear to represent specific development and expression of a unique malignant clone of cells, all of which appear to be extraordinarily homogeneous. In contrast to chronic lymphocytic leukemia, there are now data suggesting that Sizary syndrome is a monoclonal population of T cells (Table 9). Studies of peripheral blood as well as elution of T cells infiltrating the skin of patients with Sbary syndrome have included a high percentage of E-rosette-forming cells as well as similar size and surface membrane characteristics when studied in discontinuous Ficoll gradient separations323 (Table 9). Additionally, SCzary cells lack surface Ig, Fc, and C’3 receptors. Thus chronic lymphocytic leukemia appears to represent a B-cell disease and SCzary syndrome a T-cell disease. The origin of acute lymphoblastic leukemia

267

(ALL) is in doubt. Although approximately 20% of patients with ALL have cells with characteristics of T cells, as evidenced by spontaneous E rosettes, the majority of patients with ALL lack recognizable T- or B-cell markers.322.324 Indeed, there is not even conclusive evidence that such lymphoblasts are of specific lymphoid origin. Moreover, less than 2% of patients with ALL have B-cell markers.322 Yet, interestingly enough, patients with ALL possessing T-cell markers appear to have a more severe prognosis, including development of thymic masses, splenomegaly, and high blast cell counts.“‘1 Obviously, considerable work remains in attempting to isolate and identify specific receptors for specific lymphoid proliferative disorders. This work is critical because it may make it possible to identify specific viral receptors as well as to develop clinically useful antisera or methods to selectively remove these clonal populations of malignant cells. Such techniques of studying monoclonal populations of lymphoid elements can be applied to benign examples of lymphocytosis, e.g., infectious mononucleosis, in attempts to further define their specific cellular origin and the mechanism for this appearance.315 The alterations of lymphoid subpopulations in autoimmune diseases are considerably more complex because the cell populations are generally heterogeneous. The one known exception to this is the appearance of monoclonal IgD and IgM on the surface of B cells of Sjogren syndrome. R25--R2iIn contrast, studies of patients with SLE disclosed individuals with (I) marked decrease in absolute number of T cells, (2) marked decrease in absolute numbers of B cells, (3) minimal increase in absolute number of null cells, (4) decrease in percentage of T cells, (5) normal percentage of B cells, and (6) increased percentage of null cells.31”.323.32”,32X-333 Simj_ larly, some workers have reported that lymphocytes from patients with SLE respond normally others found contrary to mitogens; resu,ts 316.323.326.32X-333 Again, a possible reason for this discrepancy is the variation in response with disease activity.‘“~+326 For example, several workers have found that patients with active disease have a greater impairment in responsiveness to mitogens than patients with less active disease. Most previous studies have utilized the mitogen PHA; however, recent observations

268

ROBBINS

suggest that the con A response is a more sensitive indicator of SLE activity and associated lymphoid cell dysfunction.‘54*326 There is a strong correlation between SLE activity and impaired con A responsiveness. A lesser association is found between SLE activity and phytohemagglutinin responsiveness. In rheumatoid arthritis the situation is even more confusing because of the larger number of drugs influencing cell-mediated responses and the possibility (as with SLE) that alterations may be heavily dependent on genetic background, age, sex and disease activity. 333-33gNonetheless, it is particularly intriguing that a dramatic association of the MLC allele DW4 has now been demonstrated and confirmed in rheumatoid arthritis.34”,341 This association, suggesting genetic predisposition, may prove to be a significant clue in the pathogenesis and natural history of rheumatoid arthritis. The possible role(s) of immune complexes, autoantigens, and antilymphocyte antibodies in producing alterations of immune responsiveness are questions for further study. It has been proposed that a defect in lymphocyte subpopulations may be critical in disease pathogenesis; evidence supporting such alterations in lymphocyte subpopulations is accumulating for the SLE-like disease of New Zealand mice.“2fi A number of other alterations in lymphoid populations in disease have been demonstrated (Table 9). It is now becoming apparent that drugs may also influence surface marker expression and therefore disease pathogenesis.“‘“~““” Unlike the situation with mice, study of human living material depends in large measure on using blood, since lymph nodes, spleen, and thoracic duct are not generally available. Hence only one compartment of the lymphoid system is being characterized. Obviously, this possible bias must not be overlooked and underscores our dependence on comparable animal systems

AND GERSHWIN

for study. It is evident that extrapolation from mouse to man must be performed with caution. CONCLUSION

The interrelationships between immunity and genetics continue to strongly suggest that each of us is born with a preprogrammed set of disease predisposition, all of which may potentially be triggered by environmental exhas its posure. Indeed, perhaps immunology true birth with nihilism. Research in immunology has been proceeding at so rapid a pace that dust has yet to settle on the road. Thus the significance (or lack of it) of so many thousands of observations cannot yet be made with certainty. Future generations may look back upon our laboratory work and note with amusement that our usage of such animal models as nude, streaker, and motheaten mice, Aleutian mink, and a variety of mutants and anomalies of chickens, horses, and other animals are akin to the experiences of Dr. Doolittle and his animal entourage. Nonetheless one hopes optimistically that as the dust begins to settle on the road our experiments will grow from a “look and let’s see” approach to a definitive attempt at genetic and molecular manipulation. Indeed, it is entirely possible, if not overwhelmingly likely, that the immune system will continue to evolve and differentiate under the influence of man’s changing environment and that future generations may look upon the prevalence of autoimmunity and lymphoproliferative disease as a necessary and interesting step in the phylogenetic spectrum of immunoevolution. ACKNOWLEDGMENT The authors wish to thank Annette Mulkey, Ken Shiomoto, and Thomas Kenney for secretarial, illustrative, and editorial assistance. We also appreciated the helpful suggestions of our colleagues at the University of California, the National Institutes of Health, and the National Naval Medical Research Institute.

REFERENCES I. Glick B, Chang TS, Jaap RJ: The bursa of Fabricius and antibody production. Poult Sci 35:224-225, 1956 2. Cooper MD, Perey DY, Peterson RDA, et al: The two-component concept of the lymphoid system, in Immunologic Deficiency Diseases in Man. White Plains, National Foundation/Marchof Dimes, 1968, ~~7-14 3. Cooper MD, Peterson RDA, Good RA, et al: The functions of the thymus and bursa system in the chicken. J Exp Med 123:755102, 1966

4. Gatti RA, Stutman 0, Good RA: The lymphoid system. Annu Rev Physiol 32529 -546, 1970 5. Feldman M, Nossal GJ: Cellular basis of antibody production. Q Rev Biol47:269-362, 1972 6. Good RA: Structure-function relations in the lymphoid system, in Bach FH, Good RA (eds): Clinical Immunobiology, vol 1. New York, Academic, 1972, pp l-28 7. Gowans JL, McGregor DD: The immunological activities of lymphocytes. Prog Allergy 9: I-78, 1965

LYMPHOCYTE

SUBPOPULATIONS

8. Cooper MD, Cain WA, Van Alten PJ, et al: Development and function of the immunoglobulin-producing system. Int Arch Allergy 35:242-252, 1969 9. Cooper MD, Lawton AR, Kincade PW: A developmental approach to the biological basis for antibody diversity. Curr Top Immunobiol2:33-47, 1972 10. Warner NL: The immunological role of different lymphoid organs in the chicken. IV. Functional differences between thymic and bursal cells. Aust J Exp Biol Med Sci 431439-450, 1965 I I. Aspinall RL, Meyer RK, Graetzer MA, et al: Effect of thymectomy and bursectomy on the survival of skin homografts in chickens. J Immunol90:872-877, 1963. 12. Warner NL: The immunological role of different lymphoid organs in the chicken. II. The immunologic competence of thymic cell suspensions. Aust J Exp Biol Med Sci 42:401-416, 1964 13. Warner NL: Nature of antigen recognition site in cellular immunity. Transplant Proc 3:848-851, 1971 14. Warner NL, Szenberg A, Burnet FM: The immunological role of different lymphoid organs in the chicken. I. Dissociation of immunological responsiveness. Aust J Exp Biol Med Sci 40:373-393, 1962 15. Parrott DM, DeSousa M: Thymus-dependent and thymus-independent populations: Origin, migratory patterns and lifespan. Clin Exp Immunol8:663-684, 197 1 16. Raff MC: Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant Rev 7:52-80, 1971 17. Warner NL, Szenberg A: Immunologic studies on hormonally bursectomised and surgically thymectomised chickens: Dissociation of immunologic responsiveness, in: The Thymus in Immunobiology. New York, Plenum, 1964 18. Owen JJT: The progress and development of lymphocytes in ontogeny of acquired immunity. Ciba Foundation Symposium. Amsterdam, Excerpta Medica, 1972 19. Lawton AR, Asofsky R, Hylton MB, et al: Suppression of immunoglobulin class synthesis in mice. I. Effects of treatment with antibody to r-chain. J Exp Med 135~277-297, 1972 20. Pierce CW, Solliday SM, Asofsky R: Immune responses in vitro. Suppression of -y,, 7,. and 7, plaque forming cell responses in cultures of primed mouse spleen cells by class-specific antibody to mouse immunoglobulins. J Exp Med I35:698-7 14, 1972 21. Miller JFAP, Osoba D: Current concepts of the immunological function of the thymus. Physiol Rev 47:437-520, 1967 22. Claman HN, Chaperon EA: Immunologic complementation between thymus and marrow cells-A model for the two-cell theory of immunocompetence. Transplant Rev 1:92-113, 1969 23. Mitchell GF, Miller JFAP: Immunological activity of thymus and thoracic duct lymphocytes. Proc Nat1 Acad Sci USA 59:296-303. 1968 24. Miller JFAP, Mitchell GF: Thymus and antigenreactive ceils. Transplant Rev 1:3-42, 1969 25. Greaves MF, Janossy G: Elicitation of selective T and B lymphocyte responses by cell surface binding ligands. Transplant Rev 11:87-130, 1972 26. Claman HN, Chaperon EA, Triplett RF: Thymusmarrow cell combinations. Synergism in antibody production. Proc Sot Exp Biol 122: 1167-l 171, 1966

269

27. Miller JFAP, Basten A, Sprent T, et al: Interaction between lymphocytes in immune responses. Cell Immunol 2:469-495, 197 I 28. Feldman M, Basten A: Specific collaboration between T and B lymphocytes across a cell impermeable membrane in vitro. Nature (New Biol) 237:13-15, 1972 29. Aaskov JC, Halliday WJ: Requirement for lymphocyte-macrophage interaction in the response of mouse spleen cultures to pneumococcal polysaccharide. Cell Immunol2:335-340, 1971 30. Abdou NI, Richter M: The role of bone marrow in the immune response. Adv Immunol 12:201-270, 1970 31. Craddock CG, Longmire R, McMillan R: Lymphocytes and the immune response. N Engl J Med 285:324331,378-384, 1971 32. Dressor DW, Mitchison NA: The mechanism of immunological paralysis. Adv Immunol8:129-181, 1968 33. Gutman GA, Weissman L: Lymphoid tissue architecture: Experimental analysis of the origin and distribution of T-cells and B-cells. Immunology 23:465-479, 1972 34. DeSousa MAB, Parrott DMV, Pantelous EM: The lymphoid tissues in mice with congenital aplasia of the thymus. Clin Exp Immunol4:637-644, 1969 35. Parrott DMA, DeSousa MAB, East J: Thymus dependent areas in lymphoid organs of neonatally thymectomized mice. J Exp Med 123:191~204, 1966 36. Harris JE, Ford CE: Role of the thymus: Migration of cells from thymic grafts to lymph nodes in mice. Lancet 1:389-390, 1963 37. Cooper MD, Lawton AR, Kincade PW: A two-stage model for development of antibody-producing cells. Clin Exp Immunol 11:143-149, 1972 38. Good RA, Dalmasso AP, Martinez C, et al: The role of the thymus in development of immunologic capacity in rabbits and mice. J Exp Med 116:773-796, 1962 39. Basch RS: Immunologic competence after thymectomy. Int Arch Allergy 30: 105- 119,1966 40. Bach FH, Widmer MB, Bach ML, et al: Serologically defined and lymphocyte defined components of the major histocompatibility complex in the mouse. J Exp Med 136:1430-1444, 1972 41. Schimpl A, Wecker E: Reconstitution of a thymus cell-deprived immune system by syngeneic and allogeneic thymocytes in vitro. Eur J Immunol 1:304-306, 1971 42. Davis AJS: The thymus and the celmlar basis of immunity. Transplant Rev I:43391, 1969 43, Demant P: Genetic requirements for graft-versushost reaction in mouse. Different efficacy of incompatibility at D- and K-ends of the H-2 locus. Folia Biol (Praha) 16:273-275, 1970 44. Davies AJS, Carter RL, Leuchars E, et al: The morphology of immune reactions in normal thymectomized and reconstituted mice. III. Response to bacterial antigens: Salmonella flagella antigen and pneumococcal polysaccharide. Immunology 19:945-957, 1970 45. Davies AJS, Leuchars E, Wallis V, et al: A system for lymphocytes in the mouse. Proc R Sot Lond [Biol] 176:3699384, 197 I 46. Ackerman GA: Developmental relationship between the appearance of lymphocytes and lymphopoietic activity in the thymus and lymph nodes of the fetal cat. Anat Ret 158:387-399, 1967 47. Ackerman GA, Knouff RA: Testosterone sup-

270

ROBBINS

pression of mesenchymal lymphoepithelial

alkaline

phosphatase

nodule formation

activity and

in the bursa of Fabricius

in the embryonic chick. Anat Ret 146:23 27, 1963 48. Sata VL, Waksal

SD, Herzenberg

and separation of pre T-cells from nu/nu tion by preculture 49. Gelfand

Pyke

KW:

of human

Nature251:42ll423,

1974

T,

JM,

Albright

immunity

JF:

in culture.

in Helminths

Burnet

and dif-

immune

potential.

TC:

experimentally

precursors

Immunobiol4:13An

proteins

Lymph

New York, Academic,

Invertebrate

responses, ContempTop Cheng

FC: Lymphatics,

1970

electrophoretic

analysis

of

J lnvertebr

Pathol

81, 1969

55.

JG: Oyster

leukocytes

in infectious disease.

56. Hostetter

RK, Cooper

EL: Earthworm

immunity. Contemp Top lmmunobiol4:9 57. Lemmi CA, Cooper

immunity.

Contemp

to

Top Im-

119, 1974

Ruben LN,

ble amphibian

lymphosarcoma.

studies of a transmissi-

Cancer

book of Laboratory

Rawnsley

Med Res 14:4955522, Dardenne

JC: Neoplastic

63. Clark thymus;

SL:

III.

tiger salamanders

from a

317, 1977 HM,

Edelman

protein

GM.

Chemical

Epithelial

Il6:207

Vadehra

DV: Animal

health hazards. Anim

origin of the serum thymic

27:2999304,

of sulfate

to secretion

by the mouse

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